1. Field of the Invention
The present invention relates to a method for manufacturing gelatin with a reduced endotoxin content and to a low endotoxin gelatin.
2. Discussion of the Background
Gelatin, characterized by gelability, viscosity, foamability, adsorption-preventing capability, and the like, is a protein derived from natural substances that is employed in a variety of applications, such as foods, cosmetics, industrial products, and pharmaceuticals. In recent years, use as a cell and tissue scaffold (footing material) in regenerative treatments has also been anticipated. Cell and tissue scaffolds can be embedded as is at a site being regenerated, not only helping in the infiltration and proliferation of cells, but also playing a role in supporting cytokine as it guides cell differentiation and the like. However, use in such areas requires extremely high safety. The scaffold that is embedded is gradually degraded by proteases in the body and is controlled so as to be completely replaced by cells at the regeneration site over a period of several weeks. Since it remains within the body for an extended period in this manner, it has major effects not just on cells at the regeneration site, but on the entire body.
Normally, gelatins contain trace quantities of endotoxins. These are toxins that are present in extremely small quantities and exhibit intense heat-generating activity. Thus, when considering the use of gelatins in the realm of medical treatment, it is essential that the endotoxins be removed from the gelatin.
Endotoxins are comprised of lipopolysaccharide molecules. The molecular weight of the lipopolysaccharide subunits is said to be about 20,000. Endotoxins are deactivated by heat. However, to completely deactivate an endotoxin by heat requires heating to 250° C. for 30 minutes or more (Endotoxin Test Methods, Japanese Pharmacopoeia, 14th Ed., Revised). Known methods of deactivating endotoxins by methods other than heating include acid and base treatment methods (Japanese Unexamined Patent Publication (KOKAI) Showa No. 58-73371, which is expressly incorporated herein by reference in its entirety) in which endotoxins are degraded with an acid or a base, and methods employing acidic electrolytic water (Japanese Unexamined Patent Publication (KOKAI) No. 2004-089448, which is expressly incorporated herein by reference in its entirety). However, endotoxins are generally said to be stable relative to changes in pH.
Further, since the molecular weight of the lipopolysaccharide subunits of endotoxins is about 20,000, they cannot be removed by ordinary filtration (filtration precision: about 10 μm). Accordingly, the methods that are currently employed to remove them include distillation, reverse osmosis, and adsorption. However, the use of these methods to remove endotoxins from gelatin is difficult. Although distillation permits nearly complete removal of the endotoxins contained in water, it is not suited to removal of endotoxins from protein solutions. Reverse osmosis is incapable of passing even the amino acids constituting proteins, and is thus unsuited to filtering gelatins. Although there are adsorption methods such as applying a ZETA potential to a Nylon 66 film and filtering out the endotoxins by adsorption, in protein solutions such as gelatins, the intensity of the ions is greater than water and there are numerous factors affecting the removal rate of endotoxins, making it difficult to remove endotoxins with the same efficiency as from water (see Applied and Environmental Microbiology, Dec., p. 1375-1377 (1985), which is expressly incorporated herein by reference in its entirety).
The content of endotoxins is strictly regulated in injections administered directly into blood vessels. To remove endotoxins from such injections, neither reverse osmosis nor distillation is used; ultrafiltration is employed. In ultrafiltration, many of the lower molecules (antibiotics, salts, glucose, and the like) contained in injections can be completely passed while removing the endotoxins. In this process, the molecular weight cut-off of the ultrafiltration membrane employed is about several thousand, with a maximum of about 10,000 (Applied and Environmental Microbiology, Oct., p. 382-385 (1977), which is expressly incorporated herein by reference in its entirety; U.S. Pat. No. 4,082,737, which is expressly incorporated herein by reference in its entirety).
As set forth above, there are strict requirements for lowering the level of endotoxins in gelatin. Up to now, researchers cultivating cells and regenerative medical product manufacturers who purchase gelatins have been employing these various treatments. However, these treatments require great effort as well as being costly and time consuming. Thus, it is important to gelatin manufacturers to conduct suitable endotoxin-reducing treatments to ensure the quality of their products. However, thus far, there has been no known method for manufacturing gelatin with reduced endotoxin content that is suited to industrial production; the establishment of a method for readily manufacturing gelatin with reduced endotoxin content has become urgent.
Accordingly, an object of the present invention is to provide a method for manufacturing gelatin with reduced endotoxin content that is suited to large-quantity production. A further object of the present invention is to provide a gelatin with reduced endotoxin content.
The average molecular weight of gelatin obtained by gelling is at a minimum about 30,000 Daltons (“Da”). The use of an ultrafiltration membrane having a molecular weight cut-off of 10,000 Da to remove endotoxins contained in a solution of gelatin of such molecular weight is considered to be theoretically impossible. This is because, for example, even when processing gelatin with an average molecular weight of 30,000 Da with an ultrafiltration membrane with a molecular weight cut-off of 10,000 Da, it can be readily surmised that only the portion of ungelled gelatin with a molecular weight of 10,000 Da or less will be contained in the permeate, with the greater portion of the gelable gelatin remaining with the endotoxins in the retentate and ending up being eliminated.
In particular, the average molecular weight of commonly employed gelable gelatins is about 100,000. The removal of endotoxins by ultrafiltration membrane has been believed to be quite impossible. Further, since the molecular weight of the liposaccharide subunits of endotoxins is about 20,000, when the passing characteristics of an ultrafiltration membrane are taken into account, the reliable removal of endotoxins has been assumed to leave no margin in the selection of an ultrafiltration membrane with a molecular weight cut-off exceeding 10,000.
However, as a result of investigation conducted by the present inventors, it was discovered that in gelatin solutions, even when employing an ultrafiltration membrane with a molecular weight cut-off exceeding 10,000, the endotoxins remained in the retentate, making it possible to obtain a desired low endotoxin gelatin solution as permeate. That is, when gelatin solutions having various average molecular weights were prepared and then processed with several types of ultrafiltration membranes having molecular weight cut-offs ranging from 20,000 to 300,000 (molecular weight cut-offs permitting passage of at least a portion of the gelatin contained in the gelatin solution), the endotoxins remained in the retentate, and a gelatin solution of reduced endotoxin content was obtained as permeate.
In contrast to the low molecular weight substances (antibiotics, salts, glucose, and the like) targeted by the removal of endotoxins by conventional ultrafiltration, the present invention targets the removal of the endotoxins present in a gelatin solution. The amino acids constituting the gelatin exhibit various charge, hydrophobic, and hydrophilic properties. Thus, the state of endotoxins in the gelatin solution is thought to be one that tends to keep the endotoxins from passing through the ultrafiltration membrane. Thus, even with an ultrafiltration membrane having a molecular weight cut-off of 100,000 or 200,000, for example, which is far greater than the molecular weight of about 20,000 of the liposaccharide subunits of endotoxins, the endotoxins remain in the retentate and a desired low endotoxin gelatin solution is obtained as permeate. The present invention was devised on the basis of this completely unexpected result.